The present invention relates to the field of medical industry and is concerned with a composition of and a process for the preparation of a medicinal form of emulsions of perfluoroorganic compounds (PFOC) with gas-transporting properties, intended, in particular, for intravenous administration when compensating for blood losses and for treating various diseases accompanied by hypoxic or ischemic lesions, and also as contrast and perfusion media.
Though the range of possible applications of gas-transporting PFOC emulsions keeps on extending, on the whole the application of these preparations is still limited in view of the following factors.
The delivery of oxygen is not sufficiently effective, particularly because of insufficient oxygen capacity of PFOC emulsions compared with whole blood, which makes those engaged in developing PFOC emulsions increase the concentration of gas-transporting components, for example, up to 65 vol. % [European Patent No. 0307087B1, publication dated 08.06.1994]. However, an increase of the PFOC concentration in the emulsion is inevitably accompanied by a sharp increase in the viscosity of the preparations. As a result, emulsions with a high PFOC concentration, in spite of a high absolute oxygen capacity, do not provide sufficiently effective oxygen delivery to the tissues due to the small dynamic oxygen capacity determined by the absolute oxygen capacity/viscosity ratio. When the viscosity of the preparation is high, the compensatory reactions of the cardiovascular system, directed to increasing the minute volume and raising the rheological properties of blood, can provide neither sufficient perfusion of large vessels nor effective circulation, and, consequently, the delivery of oxygen to the tissues is disturbed.
The reactogenicity of PFOC emulsions is relatively high. The reactogenicity is associated, in the first place, with the formation of peroxide radicals in the stabilizing agent [L. E. McCoy, C. A. Berker, T. H. Goodin, M. J. Barnhart, //Scann. Electron. Microscopy, 1984, v. 1, p. 311] which forms an adsorption layer of the PFOC particles, and, in the second place, with the presence of large particles in the emulsion, which are recognized by the immune system of the organism as foreign agents [S. I. Vorob""ev et al., xe2x80x9cComparative study of some perfluorocarbon emulsionsxe2x80x9d, in: Physiological Activity of Fluorine-containing Compounds (Experiment and Clinic), Pushchino, 1995, pp. 33-41 (in Russian)].
The possibility of manifestation of toxicity of the emulsions, caused by the effect of lipophilic perfluorocarbons rapidly eliminable from the organism, traditionally used for producing gas-transporting blood substitutes, on biological structures, and by possible aggregation and coarsening of emulsion grains in the blood flow. Rapidly eliminable perfluorocarbons easily interact with enzymatic systems and biological membranes, formed elements and proteins of blood, and, as is shown by a number of investigations, Induce an anaphilactogenic reaction and delayed death (in 50-80 days) of large laboratory animals having a low blood flow rate, comparable with that in humans (A. N. Sklifas, V. V. Obraztsov, K. N. Makarov, D. G. Shekhtman, N. I. Kukushkin, xe2x80x9cInvestigation of the Toxicity Mechanism of Perfluorodecalin Emulsion for Rabbitsxe2x80x9d, in collected articles: xe2x80x9cPerfluorocarbon Active Media for Medicine and Biology. New Aspects of Researchxe2x80x9d. Pushchino, 1993, pp. 129-135 (in Russian)].
High cost of PFOC emulsion preparations, associated with the necessity of preparing individual PFOCs and high requirements to their purification from admixtures, which makes them difficult of access for users.
Binary PFOC emulsions are known (see, e.g., USSR Patent No. 797546, publ. in Bull. xe2x80x9cOtkrytiya, Izobreteniya, . . . xe2x80x9d, No. 2, 15.01.1981, or RF Patent No. 2088217, publ. in Bull. xe2x80x9cIzo-breteniya . . . (Zayavki . . . ,)xe2x80x9d, No. 24, 27.08.1997), in which, for lowering the toxicity, in addition to rapidly eliminable perfluorocarbons, slowly eliminable substances are contained, for instance, perfluorinated tertiary amines, which possess expressed lipophobic properties and do not interact in any manner with biological objects. For example, it has been shown that the presence in the composition of the emulsions and even long-term acctmulation in organs of slowly eliminable perfluorotributylamine, which is retained in macrophages of different organs for a period commensurable with the life time of animals (and which is therefore not used for medical purposes) does not bring about any pathological changes. This is confirmed by more than 30 years of investigations aimed at finding toxicity and probable cancerogenicity and by 16 years of experience in medical uses of emulsions of perfluoroorganic compounds, containing other relatively slowly eliminable perfluorinated tertiary amines [Biomaterials, artificial cells, and artificial organs. 1988, vol. 16, No. 1-3, Special Issue from III International Symposium on xe2x80x9cBlood Substitutesxe2x80x9d; A. M. Golubev, Advances in and Prospects of Studying the Influence of Fluorocarbon Blood Substitutes on Biological Systems./Perfluorocarbon Active Media for Medicine and Biology (New Aspects of Research). Pushchino: ONTI of Pushchino Research Center, Russian Academy of Sciences, 1993, pp. 88-93 (in Russian)].
The binary PFOC emulsion according to RF Patent No. 2088217 is the closest to the claimed emulsion with regard to the formulation of its components. It comprises a mixture of a rapidly eliminable lipophilic perfluorocarbon perfluorodecalin (PFD) or perfluorooctylbromide and a slowly eliminable lipophobic perfluorinated tertiary amine perfluoro-N-(4-methylcyclohexyl)-piperidine (PFMCP) in a ratio of 40-50 vol. % to 1-10 vol. %, and is stabilized by phospholipids the amount of which is from 2 to 6%. Upon intravenous administration of such emulsion, 100% of rabbits survive during 60 days of observations. However, this emulsion has a high viscosity (14-16 cP), which, as indicated above, in spite of the high absolute oxygen capacity of the preparation, leads to a sharp drop of the dynamic oxygen capacity and does not provide efficient delivery of oxygen.
Furthermore, the use of such sharply differing in their physical properties PFOC as the lipophilic PFD and lipophobic PFMCP leads to clustering of the fluorocarbon phase inside the emulsion particles, which complicates the choice of the stabilizing agent and impairs the emulsion stability. As a result, the emulsion dispersity changes both during storage and when the emulsion enters the blood flow.
Finally, in this invention individual highly purified perfluorodecalin and perfluoro-N-(4-methycyclohexyl)-piperidine are used for preparing the emulsion, which, as already indicated, makes the PFOC emulsion appreciably more expensive.
A PFOC emulsion is known, which is closest to the claimed one in the composition and in the ratio of the components [see RF Patent No. 2070033, publ. in Bull. xe2x80x9cIzbreteniya . . . (Zayavki, . . . )xe2x80x9d, No. 34, 27.08.97]. This emulsion contains perfluorocarbon in the form of cis- and trans-isomers of perfluorodecalin and perfluorinated tertiary amine perfluoro-N-(4-methylcyclohexyl)-piperidine in lower concentrations of both components: 7 vol. % and 3.5 vol. %, respectively, and it is stabilized by a polyoxyethylene-polyoxypropylene copolymer with a molecular mass of 6-8 thousand Dalton (Da) with a mean size of emulsion particles of 0.08-0.1 xcexcm. This composition, with a comparatively small value of the absolute oxygen capacity (7 vol. % of oxygen at pO2 of 760 mm Hg), ensures owing to its small viscosity (3.5 cP) a higher dynamic oxygen capacity than other known PFOC emulsions (see Table 1 in the specification to said RF Patent), i.e., it ensures better oxygen delivery to the tissues. Owing to the high proportion of the slowly eliminable PFMCP (the PFMCP/PFD ratio=1:2) in the PFOC formulation and the high degree of monodispersity of the particles along with their size, this emulsion is not toxic for large animals. However, the emulsion according to RF Patent No. 2070033 is not free from a number of disadvantages: a relatively high reactogenicity because of formation of peroxide compounds in the stabilizing agent in the process of manufacture and during storage of the emulsion; insufficient stability upon repeated freezing-defrosting the emulsion, when the emulsion enters the blood flow, and when it gets in contact with high-molecular dextrans, since two PFOC sharply differing in their physicochemical properties are used in the composition of the emulsion; and a high cost of the preparation due to the use of highly purified PFD and PFMCP.
Known in the art is a process for the preparation of PFOC emulsions for medical purposes, in which process for reactogenicity it is proposed to decrease the mean size and to increase the monodispersity of the emulsion particles (see the specification to RF Patent No. 2070033). These properties are secured by adding dropwise a mixture of two forms of liquid PFOC to an aqueous solution of a stabilizing agent, thereby precluding the origination of a macroscopic interface, increasing the contact time and area of the PFOC with the stabilizing agent in the step of preparing the pro-emulsion. A submicron emulsion is prepared in a two-circuit homogenization system in the process of 12-fold recirculation in the homogenizer circuits. According to the specification of the cited invention, the first and second circuits of the homogenizer are used alternately, the homogenization process being thus slowed down, because returning from the second circuit to the first circuit leads to coarse pro-emulsion particles and even drops of PFOC, which are inevitably formed and retained on the walls of the chamber and pipes in the first circuit, getting into the already finely divided fine-dispersed emulsion. Furthermore, as it was already mentioned when criticizing the formulation of the emulsion, this process does not prevent formation of peroxide compounds.
It is an object of the proposed invention to provide such a composition of a PFOC emulsion, which ensures a reduction of the reactogenicity of the preparation and of the toxicity thereof for large animals and humans, a high dynamic oxygen capacity, a high stability on repeated freezing-defrosting and on contact with high-molecular dextrans used as blood substitutes.
Another object of the proposed invention is to develop such a process for the preparation of a pro-emulsion and a submicron emulsion proper, which, while preserving a high monodispersity of the system and with a prescribed small size of particles, inhibits the formation of peroxide compounds in the course of preparing the emulsion and during storage thereof.
Still another object of the invention is to reduce the capital inputs, to simplify and speed-up the process of preparing the PFOC emulsion, this being necessary for the industrial-scale manufacture of the drug.
The first object of the present invention is accomplished by that the known PFOC emulsion for medical purposes, which comprices a rapidly eliminable perfluorocarbon and a slowly eliminable perfluorinated tertiary amine, a stabilizing agent and a physiologically acceptable aqueous-saline solution with an energy-metabolism substrate, according to the invention, further comprises admixtures of at least three rapidly eliminable cis- and trans-isomers of perfluorocarbons C7-C10 and of at least three slowly eliminable perfluorinated tertiary amines C11-C13, close in physicochemical properties to the main perfluoroorganic compounds, for instance, in the critical temperature of dissolution in hexane, and minor admixtures of H-perfluoroalkanes. The content of the admixtures of rapidly eliminable perfluorocarbons does not exceed 15% of the content of the main perfluorocarbon; the content of the admixtures of slowly eliminable perfluorinated tertiary amines does not exceed 50% of the content of the main perfluorinated tertiary amine; and the content of admixtures of H-perfluoroalkanes does not exceed 0.1 vol. %. A the rapidly eliminable perfluorocarbon a mixture of cis- and trans-isomers of perfluorodecalin or perfluorooctyl bromide is used, and as the slowly eliminable PFOC perflluoro-N-4-(methylcyclohexyl)-piperidine is used. As the stabilizing agent a polyoxyethylene-polyoxypropylene copolymer with the molecular mass of 6-10 thousand Da is used.
The ratio of the perfluorocarbons and perfluorinated tertiary amires is 2:1 or 3:1.
In the preferable embodiment the emulsion comprises the main rapidly eliminable perfluorocarbon in the form of cis- and trans-isomers of perfluorodecalin in an amount of 6 vol. % and admixtures of rapidly eliminable perflufluorocarbons C7-C10 comprising a mixture of perflufluoromethylindane, perfluoro-1-methyl-3-propylcyclohexane, trans-perfluoroindane, perfluoro-4-oxodecalin, perfluorobutylcyclohexane, perfluoropropylcyclohexane, perfluoroethylcyclohexane, perfluorobutylcyclopentane, cis-perfluoro-1-methyl-2-ethylcyclohexane in a total amount of 0.7 vol. %; the main slowly eliminable perfluorinated tertiary amine perfluoro-N-4-(methylcyclohexyl)-piperidine in the form of a mixture of isomers in an amount of 2.3 vol. % and an admixture of slowly eliminable perfluorinated tertiary amines C11-C13 comprising a mixture of perfluoro-N-(4-methylcyclohexyl)-2-methylpyrrolidine, perfluoromethylbutyl-(4-methylcyclohexyl)-amine, cis- and trans-isomers of perfluoromethylpropyl-(4-methylcyclohexyl)-amine, isomers of perfluoromethylpropyl-(methylcyclopentyl)-amine and perfluoro-N-(4-methylcyclohexyl)-1-methylpiperidine in a total amount of 1.0 vol. %; and also admixtures of H-perfluoroalkanes in an amount of 0.02 vol. %.
In the preferred embodiment the ratio of the rapidly and slowly eliminable PEOC is 2:1. The content of the polyoxyethylene-polyoxypropylene copolymer with a molecular mass of 8 thousand Da is 4%, with the ratio of the polyoxyethylene and polyoxypropylene blocks in the copolymer being 4:1.
When the PFOC emulsion is used as a plasma substitute and for the perfusion of organs, the physiologically acceptable aqueous-saline solution contains 102 mM of NaCl, 5.2 mM of KCl, 1.8 mM of MgCl2, 7.7 mM NaHCO3, 1.65 mM of NaH2PO4 and 11 mM of D-glucose.
When the emulsion is used for the anti-ischemic protection of the heart disconnected from the blood flow in the course of pharmaco-ice-chip cardioplegia, the aqueous-saline solution contains 102 mM of NaCl, 5.2 mM of KCl, 1.8 mM of MgCl2, 7.7 mM NaHCO3, 1.65 mM of NaH2PO4 and 11 mM of D-glucose, 5 mM of sodium pyruvate, 5 mM of sodium xcex2-oxybutyrate, 5 mM of sodium succinate, 5 mM of sodium glutamate, 5 mM of taurine.
Such content and composition of perfluorocarbons and perfluorinated tertiary amines corresponds to the composition of underrefined PFD and PFMCP which practically do not display any toxic properties either per se (in tests on isolated cultivated lymphoid cells) or in the formulation of the emulsion (in tests on small rodents and rodents), similarly to individual highly purified PFD and PFMCP containing no admixtures of other PFOC (Tables 1 and 2). This should be accounted by that the toxicity of PFOC stems not from their form, but from the presence of underfluorinated admixtures. If the latter are absent, then the next factors responsible for the toxicity are the concentration of fluorine in the aqueous phase, the detergent properties of the stabilizing agent, the appearance of peroxides and the presence of coarse or easily coalescing particles in the emulsion. Death of large animals, e.g., of rabbits, can be caused, as it follows from the data presented in Table 3 of the specification to RF Patent 2088217, by the use in the composition of the emulsion of exclusively rapidly eliminable highly lipophilic perfluoroorganic compounds, whereas introducing a lipophobic, i.e., a slowly eliminable PFMCP component, leads to a decrease in the toxicity, as a result of which the survival of rabbits reaches 100% during 60 days after administering 20 ml of the PFOC emulsion per kg of the body weight. A comparison of PFOC emulsions in terms of this parameter shows (Table 2) that the composition being patented by us does not cause death of rabbits even after 20-fold administration of the indicated dose. More than that, if the composition being patented is administered prior to, simultaneously with, or at least a weak after the administration of an emulsion manufactured only from PFD (ensuring 100% death of rabbits), all the animals survive (Table 2).
Impurity PFOCs present in the formulation of the emulsion have a structure close to the structure of the main PFOCs and constitute a number of PFOCs with gradually changing lipophilic-lipophobic properties (FIG. 1, Table 3). This contributes to the formation of a more homogeneous not clustered fluorocarbon phase inside the emulsion particles, increases the stability of the emulsion particles, decreases the intramolecular distillation phenomena responsible for coarsening of the particles, inhibits the aggregation of the particles both when they enter the blood flow and upon repeated freezing and upon contact with high-molecular oncotic agents of dextran nature (Table 4).
The presence in the fluorocarbon phase of a set of PFOCs with manifest lipophobic properties, particularly of perfluorinated tertiary amines displaced to the surface of the fluorocarbon; phase, contributes to better interaction with the stabilizing agent, i.e., with a polyoxyethylene-polyoxypropylene block copolymer, and to retaining it in the adsorption layer. As a result, the surface of the emulsion particles becomes less loose, acquires improved rheological characteristics: a lower viscosity and, as a consequence, a higher dynamic oxygen capacity at the same value of the absolute oxygen capacity, as well as an increased stability both in vitro (Table 4) and in the blood flow, which manifests itself in an increased time of the emulsion circulation (Table 5).
In the known literature sources no description can be found of PFOC emulsions comprising a mixture of several PFOC in structure, which differ in the physicochemical parameters so that they constitute a series of compounds with the properties gradually changing from lipophilic to lipophilic ones.
In FRG laid-open Application No. 4325100 A1 a PFOC emulsion is described, containing admixtures of H-perfluoroalkanes in an amount of 1 to 5%. The authors of said Application believe that this makes it possible to improve the oxygen-transporting properties of the emulsion and to lower its cost by reducing expenditures for the PFOC purification. However, an analysis carried out by us has shown that the content of H-perfluoroalkanes higher than 1 wt. % (0.5 vol. %) can lead to the appearance of toxicity in the preparations of emulsions both for isolated cultivated cells and for an integral organism.
The composition of the PFOC determines the choice of a particular stabilizing agent.
At present for the preparation of emulsions of rapidly eliminable PFOCs expensive phospholipids of organic origin are used, that have a high affinity for lipophilic PFOCs and require antioxidant additives for preventing peroxide oxidation. In the case of preparing binary compositions from two PFOCs relatively close in lipophilic-lipophobic properties (perfluorodecalin and perfluorotripropylamine), a mixture of phospholipids with a polyoxyethylene-polyoxypropylene copolymer is used [USSR Patent No. 797546, belongs to Green Cross Corp.]. A synthetic cheap polyoxyethylene-polyoxypropylene copolymer is used for stabilizing emulsions containing lipophobic PFOCs. Such stabilizers are, for instance, perfluorobutylamine which is not eliminable from the organism of animals and therefore is used only in experimental pharmacology and physiology, or PFMCP and, less successfully, mixtures of PFD with PFMCP. The PFOC composition proposed in the present invention, consisting of a mixture of several PFOCs with gradually changing lipophilic-lipophobic properties is also adequately stabilized in an emulsified form by a polyoxyethylene-polyoxypropylene copolymer, which makes the emulsion essentially cheaper and allows taking simple measures for minimizing the danger of the origination of hydroperoxides in the course of manufacturing and storing the emulsion, without additionally introducing antioxidants into the formulation.
The second object of the present invention is accomplished by that in the known process for the preparation of a PFOC emulsion, which comprises mixing depyrogenized components by passing liquid PFOCs through an aqueous solution of a stabilizing agent and multiple homogenizing of the resultant mixture in a two-circuit high-pressure homogenizer, according to the invention, before mixing the components, a mixture of liquid PFOCs and an aqueous solution of a stabilizing agent are saturated with carbon dioxide gas, after that the aqueous solution of the stabilizing agent is heated at a temperature not exceeding 75xc2x0 C., then the mixture of liquid PFOCs is introduced in several jets into the cooled aqueous solution of the stabilizing agent under intensive stirring and feeding carbon dioxide gas, simultaneously passing the resulting coarse-dispersed pro-emulsion several times through the first circuit of the homogenizer, whereafter the finely divided pro-emulsion is subjected to homogenization in the second circuit of the homogenizer till the required dispersity is obtained with feeding carbon dioxide gas, and an aqueous-saline composition is added. The obtained medicinal form is poured into containers.
Saturating the components to be mixed with carbon dioxide gas makes it possible to provide conditions under which in the course of manufacturing the emulsion the formation of peroxide compounds is minimized, the presence of which, as well as the presence of coarse particles, brings about the origination of reactogenicity of emulsions (Table 5). Under experimental conditions the presence of reactogenicity was judged from the drop in the content of neutrophilic leukocytes in the peripheral blood of the rabbit after administering the emulsion in the dose of 10 ml per kg of the body weight. Quantitatively the degree of reactogenicity was calculated from the value of the neuroleptic index: an increase of the neuroleptic index by more than 3 units is indicative of a noticeable reactogenicity of the emulsions [M. V. Berkos, Emulsions of Perfluorocarboh Compounds in Experimental Intravenous Administration. Abstract of Candidate""s Thesis, Leningrad, 1991].
Warming-up of the aqueous solution of the stabilizing agent (proxanol) makes it possible to depyrogenize the solution without passing it through sorbents which impair the quality of the stabilizing agent and change its molecular-mass distribution and the surface-active properties. Deviations from the indicated temperature values impair the surface-active properties of the stabilizing agent or increase the content of hydroperoxides in it.
Jet-like introducing of liquid PFOCs into the aqueous solution of the stabilizing agent with intensive stirring and simultaneous passing the resulting mixture through the high-pressure homogenizer contributes to speeding-up the process of preparing the pro-emulsion. The rate of PFOC feeding, stirring and passing the mixture through the homogenizer is regulated in such a manner that the formation of the macroscopic PFOC/water interface is precluded, this being a prerequisite for obtaining a monodisperse emulsion.
Owing to sequential use of two homogenizer circuits, the number of recirculation cycles, compared with the prototype, is reduced from 12 to 9.
Thus, the proposed process makes it possible to obtain large lots of emulsions, to minimize the formation of peroxide compounds, while preserving a high monodispersity of the emulsion particles on the level of 0.05-0.1 xcexcm (Table 6).
The use of a mixture of underrefined perfluorocarbons and perfluorinated tertiary amines instead of individual highly purified PFOCs, as well as a simpler and more rapid technology provides a possibility to accomplish the third object of the invention: to cut down expenses for manufacturing the emulsion and to reduce the cost of the preparation at least 2- or 5-fold, simultaneously enhancing the stability and improving the rheological properties of the emulsions. Thereby prospects are opened for industrial manufacturing and wider uses of PFOC emulsions in clinical practice.